BACKGROUND: Hyperthermia (HT), while a cancer treatment approach, isn't always effective alone. Therefore, identifying hyperthermia enhancers is crucial. We demonstrated that Mito-TEMPO ([2-[(1-Hydroxy-2,2,6,6-tetramethylpiperidin-4-yl) amino]-2-oxoethyl]-triphenylphosphanium, MT) acts as a potent thermosensitizer, promoting cell death in human cervical cancer (HeLa) cells. METHODS: Cells were pretreated with 0.4 mM MT for 5 minutes, followed by exposure to hyperthermia (42 °C for 60 minutes). The impacts of MT/HT on cell viability, proliferation, apoptosis, endoplasmic reticulum (ER) stress, apoptosis-related proteins and autophagy, autophagy-related proteins expression were measured. The relationships between autophagy and apoptosis were further investigated using the specific autophagy inhibitor chloroquine (CQ) and the autophagy inducer rapamycin (Rapa). RESULTS: The combined treatment reduced the mitochondrial membrane potential (MMP) and increased ROS production. It also upregulated the pro-apoptotic protein Bax and downregulated anti-apoptotic proteins such as Bcl-2 and MCL-1. As a result, Caspase-3 was activated. Additionally, the combined treatment upregulated the expression of p-PERK/PERK, ATF-4, CHOP proteins. Moreover, the combined treatment also increased the expression of LC3 II and p62, decreased expression of LAMP 1 and Cathepsin D and increased lysosomal pH, indicating coordinated changes in autophagy regulation. Notably, intensification of apoptosis induced by the combined treatment was observed with CQ, whereas attenuation was seen with Rapa. CONCLUSIONS MT effectively enhanced HT-induced apoptosis in HeLa cells. Elevated ER stress and interruption of autophagy flux are the possible underlying molecular mechanisms for this phenomenon. These findings suggested MT can act as a potential thermosensitizer, highlighting its versatility in cancer treatment strategies.
Humans ; Apoptosis/drug effects* ; Autophagy/drug effects* ; HeLa Cells ; Uterine Cervical Neoplasms/therapy* ; Female ; Hyperthermia, Induced ; Spin Labels ; Endoplasmic Reticulum Stress/drug effects* ; Cyclic N-Oxides/pharmacology* ; Cell Survival/drug effects*

OBJECTIVES: To investigate the regulatory effects of Aurora-A in regulating proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of cervical cancer cells and the role of actin-related protein 2/3 complex subunit 4 (ARPC4) in mediating its effects. METHODS: The plasmids pCDH-NC, pCDH-Aurora-A, and shRNA-ARPC4 were used for inducing Aurora-A overexpression or ARPC4 knockdown in HeLa cells. The cells were divided into vector group, Aurora-A overexpression group, Aurora-A overexpression+ARPC4 knockdown group, and Aurora-A overexpression+NF‑κBp65 inhibitor group and transfected with the corresponding plasmids. The proliferation, colony-forming ability, migration and invasion of the treated Hela cells was evaluated using EdU immunofluorescence assay, crystal violet staining, scratch assay, Transwell assay, and Matrigel assay. Western blotting was performed to detect the changes in cellular expressions of EMT-related proteins and expression levels of NF-κBp65 and ARPC4. RESULTS: The expression of ARPC4 was significantly decreased in HeLa cells with Aurora-A knockdown and increased in Aurora-A-overexpressing cells. Aurora-A overexpression obviously promoted proliferation, migration, and invasion abilities of HeLa cells, and these effects was significantly antagonized by ARPC4 knockdown. In Aurora-A-overexpressing cells, the phosphorylation level of NF-κBp65 and the expression level of ARPC4 were increased significantly, and application of the NF‑κBp65 inhibitor obviously lowered the expression level of ARPC4. CONCLUSIONS Aurora-A overexpression upregulates the expression of ARPC4 by activating the NF-κBp65 signaling pathway, thereby promoting migration, invasion and EMT of HeLa cells.
Humans ; Uterine Cervical Neoplasms/metabolism* ; Female ; HeLa Cells ; Epithelial-Mesenchymal Transition ; Signal Transduction ; Cell Movement ; Neoplasm Invasiveness ; Cell Proliferation ; Aurora Kinase A/metabolism* ; Transcription Factor RelA/metabolism* ; Neoplasm Metastasis

(±)-Penicithrones A-D (1a/1b-4a/4b), four novel pairs of anthrone-cyclopentenone heterodimers characterized by a distinctive bridged 6/6/6-5 tetracyclic core skeleton, together with three previously identified compounds (5-7), were isolated from the crude extract of the mangrove-derived fungus Penicillium sp., guided by heteronuclear single quantum correlation (HSQC)-based small molecule accurate recognition technology (SMART 2.0) and liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based molecular networking. The structural elucidation of new compounds was accomplished through comprehensive spectroscopic analysis, and their absolute configurations were determined using DP4+ ¹³C nuclear magnetic resonance (NMR) calculations and electronic circular dichroism (ECD) calculations. Compounds 1a/1b-4a/4b demonstrated moderate cytotoxicity against three human cancer cell lines HeLa, HCT116 and MCF-7 with half maximal inhibitory concentration (IC₅₀) values ranging from 15.95 ± 1.64 to 28.56 ± 2.59 μmol·L^-1.
Humans ; Penicillium/chemistry* ; Molecular Structure ; Cyclopentanes/isolation & purification* ; Cell Line, Tumor ; Antineoplastic Agents/pharmacology* ; Tandem Mass Spectrometry ; Dimerization ; HeLa Cells ; Magnetic Resonance Spectroscopy

The development of safe and effective carriers is crucial for improving the in vivo stability of siRNA drugs and facilitating their clinical translation. Chitosan (CS), a natural cationic polymer, shows great potential in nucleic acid drug delivery. To optimize the physicochemical properties of CS/siRNA nanoparticles (NPs) and increase their siRNA delivery efficacy, in this study, hyaluronic acid (HA) was added into CS to form stable complex NPs through electrostatic interactions. The HA component is able to target the CD44 receptors on the surface of tumor cells, facilitating efficient siRNA delivery. First, we systematically investigated the effects of the molecular weights and mass ratio of CS and HA on the physicochemical properties of CS/HA NPs. The results showed that at HA: CS mass ratios of approximately 5:5 and 6:4, the complex NPs exhibited small particle sizes, narrow size distribution, and high storage stability. Under similar conditions, the size of CS/HA NPs increased with the increase in the molecular weights of CS and HA. Based on these findings, suitable conditions were selected to prepare CS/HA NPs for siRNA delivery. Cell experiments demonstrated that the introduction of HA effectively reduced the cytotoxicity of the CS delivery system and enhanced the NP uptake. The CS/HA/siRNA NPs achieved 50% to 60% silencing of the luciferase gene in HeLa-Luc cells. CS/HA NPs formed smaller nanoparticles with siRNA than pure CS and mediated specific interactions with tumor cells via HA, leading to efficient siRNA delivery. These findings provide valuable insights into the construction of natural polymer composite nanoparticles for application in siRNA delivery.
Hyaluronic Acid/chemistry* ; Chitosan/chemistry* ; RNA, Small Interfering/administration & dosage* ; Nanoparticles/chemistry* ; Humans ; Particle Size ; HeLa Cells ; Hyaluronan Receptors

Conventional cancer therapies, such as radiotherapy and chemotherapy, often damage normal cells and may induce new tumors. Oncolytic viruses (OVs) selectively target tumor cells while sparing normal cells. Most OVs used in clinical trials have been genetically engineered to enhance their ability to target tumor cells and activate immune responses. To develop a specific OV-based approach for treating cervical cancer, this study constructed an oncolytic adenovirus that delivered a base editor targeting oncogenes to achieve efficient killing of tumor cells through inhibiting tumor growth and directly lysing tumor cells. We utilized the human telomerase reverse transcriptase (TERT) promoter to drive the expression of adenovirus early region 1A (E1A) and successfully constructed the P-hTERT-E1A-GFP vector, which was validated for its activity in cervical cancer cells. Given the critical role of the MYC oncogene in the research of oncology, identifying efficient editing sites for the MYC oncogene is a key step in this study.Three MYC-targeting gRNAs were engineered and co-delivered with ABE8e base editor plasmids into HEK293T cells. Following puromycin selection, Sanger sequencing demonstrated differential editing efficiencies: MYC-1 (43%), MYC-2 (25%), and MYC-3 (35%), identifying MYC-1 as the most efficient editing locus. By constructing the P-ABEs-hTERT-E1A-GFP and P-MYC gRNA-hTERT-E1A-GFP vectors, we successfully packaged the virus and confirmed its specificity and efficacy. The experimental results demonstrate that this novel oncolytic adenovirus effectively inhibits the growth of HeLa cells in vitro, providing new experimental evidence and potential strategies for treating cervical cancer based on the HeLa cell model.
Humans ; Uterine Cervical Neoplasms/pathology* ; Oncolytic Viruses/genetics* ; Female ; HEK293 Cells ; Oncolytic Virotherapy/methods* ; Adenoviridae/genetics* ; Gene Editing/methods* ; Telomerase/genetics* ; Adenovirus E1A Proteins/genetics* ; Genetic Vectors/genetics* ; HeLa Cells

N⁶-methyladenosine (m⁶A) modification plays a critical role in cell cycle regulation, while the mechanism of m⁶A in regulating mitosis remains underexplored. Here, we found that the total m⁶A modification level in cells increased during mitosis by the liquid chromatography-mass spectrometry/mass spectrometry and m⁶A dot blot assays. Silencing methyltransferase-like 3 (METTL3) or METTL14 results in delayed mitosis, abnormal spindle assembly, and chromosome segregation defects by the immunofluorescence. By analyzing transcriptome-wide m⁶A targets in HeLa cells, we identified polo-like kinase 1 (PLK1) as a key gene modified by m⁶A in regulating mitosis. Specifically, through immunoblotting and RNA pulldown, m⁶A modification inhibits PLK1 translation via YTH N⁶-methyladenosine RNA binding protein 1, thus mediating cell cycle homeostasis. Demethylation of PLK1 mRNA leads to significant mitotic abnormalities. These findings highlight the critical role of m⁶A in regulating mitosis and the potential of m⁶A as a therapeutic target in proliferative diseases such as cancer.
Humans ; Polo-Like Kinase 1 ; Cell Cycle Proteins/metabolism* ; Proto-Oncogene Proteins/metabolism* ; Protein Serine-Threonine Kinases/metabolism* ; Mitosis/physiology* ; HeLa Cells ; Adenosine/genetics* ; Methyltransferases/metabolism* ; RNA, Messenger/metabolism* ; RNA-Binding Proteins/metabolism*

OBJECTIVE: To investigate the effects of asperuloside on cervical cancer based on endoplasmic reticulum (ER) stress and mitochondrial pathway. METHODS: Different doses (12.5-800 µg/mL) of asperuloside were used to treat cervical cancer cell lines Hela and CaSki to calculate the half maximal inhibitory concentration (IC₅₀) of asperuloside. The cell proliferation was analyzed by clone formation assay. Cell apoptosis, intracellular reactive oxygen species (ROS) and mitochondrial membrane potential were determined by flow cytometry. The protein expressions of cleaved-caspase-3, Bcl-2, Bax, Cyt-c, cleaved-caspase-4 and glucose-regulated protein 78 (GRP78) were analyzed by Western blot. And the inhibitor of ER stress, 4-phenyl butyric acid (4-PBA) was used to treat cervical cancer cells to further verify the role of ER stress in the apoptosis of cervical cancer cells induced by asperuloside. RESULTS: Asperuloside of 325, 650, and 1300 µg/mL significantly inhibited the proliferation and promoted apoptosis of Hela and CaSki cells (P<0.01). All doses of asperuloside significantly increased intracellular ROS levels, reduced mitochondrial membrane potential, significantly reduced Bcl-2 protein expression level, and increased Bax, Cyt-c, GRP78 and cleaved-caspase-4 expressions (P<0.01). In addition, 10 mmol/L 4-PBA treatment significantly promoted cell proliferation and reduced apoptosis (P<0.05), and 650 µg/mL asperuloside could reverse 4-PBA-induced increased cell proliferation, decreased apoptosis and cleaved-caspase-3, -4 and GRP78 protein expressions (P<0.05). CONCLUSION Our study revealed the role of asperuloside in cervical cancer, suggesting that asperuloside promotes apoptosis of cervical cancer cells through ER stress-mitochondrial pathway.
Female ; Humans ; Uterine Cervical Neoplasms/metabolism* ; Caspase 3/metabolism* ; bcl-2-Associated X Protein/metabolism* ; Reactive Oxygen Species/metabolism* ; Endoplasmic Reticulum Chaperone BiP ; HeLa Cells ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; Apoptosis ; Endoplasmic Reticulum Stress ; Cell Line, Tumor

The purpose of this study is to construct a muscle-specific synthetic promoter library, screen out muscle-specific promoters with high activity, analyze the relationship between element composition and activity of highly active promoters, and provide a theoretical basis for artificial synthesis of promoters. In this study, 19 promoter fragments derived from muscle-specific elements, conserved elements, and viral regulatory sequences were selected and randomLy connected to construct a muscle-specific synthetic promoter library. The luciferase plasmids pCMV-Luc and pSPs-Luc were constructed and transfected into the myoblast cell line C2C12. The activities of the synthesized promoters were evaluated by the luciferase activity assay. Two non-muscle-derived cell lines HeLa and 3T3 were used to verify the muscle specificity of the highly active promoters. The sequences of promoters with high activity, good muscle specificity, and correct sequences were analyzed to explore the relationship between the element composition and activity of promoters. We successfully constructed a muscle-specific promoter library and screened out 321 effective synthetic promoter plasmids. Among them, the activity of SP-301 promoter was 5.63 times that of CMV. The 15 promoters with high activity were muscle-specific. In the promoters with high activity and correct sequences, there was a relationship between their element composition and activity. Muscle-specific elements accounted for a high proportion in the promoters, while they had weak correlations with the promoter activity, being tissue-specific determinants. Viral elements accounted for no less than 20% in highly active promoters, which may be the key elements for the promoter activity. The content of conserved elements was proportional to the promoter activity. This study lays a theoretical foundation for the synthesis of tissue-specific efficient promoters and provides a new idea for the construction and application of in-situ gene delivery systems.
Promoter Regions, Genetic ; Humans ; Animals ; Mice ; Gene Library ; Cell Line ; Transfection ; HeLa Cells ; Luciferases/metabolism* ; Muscle, Skeletal/metabolism* ; Plasmids/genetics* ; Myoblasts/cytology*

BACKGROUND: Long non-coding RNA colon cancer-associated transcript 1 (CCAT1) is involved in transforming multiple cancers into malignant cancer types. Previous studies underlining the mechanisms of the functions of CCAT1 primarily focused on its decoy for miRNAs (micro RNAs). However, the regulatory mechanism of CCAT1-protein interaction associated with tumor metastasis is still largely unknown. The present study aimed to identify proteome-wide CCAT1 partners and explored the CCAT1-protein interaction mediated tumor metastasis. METHODS: CCAT1-proteins complexes were purified and identified using RNA antisense purification coupled with the mass spectrometry (RAP-MS) method. The database for annotation, visualization, and integrated discovery and database for eukaryotic RNA binding proteins (EuRBPDB) websites were used to bioinformatic analyzing CCAT1 binding proteins. RNA pull-down and RNA immunoprecipitation were used to validate CCAT1-Vimentin interaction. Transwell assay was used to evaluate the migration and invasion abilities of HeLa cells. RESULTS: RAP-MS method worked well by culturing cells with nucleoside analog 4-thiouridine, and cross-linking was performed using 365 nm wavelength ultraviolet. There were 631 proteins identified, out of which about 60% were RNA binding proteins recorded by the EuRBPDB database. Vimentin was one of the CCAT1 binding proteins and participated in the tumor metastasis pathway. Knocked down vimetin ( VIM ) and rescued the downregulation by overexpressing CCAT1 demonstrated that CCAT1 could enhance tumor migration and invasion abilities by stabilizing Vimentin protein. CONCLUSION CCAT1 may bind with and stabilize Vimentin protein, thus enhancing cancer cell migration and invasion abilities.
Humans ; HeLa Cells ; RNA, Long Noncoding/metabolism* ; Cell Line, Tumor ; Cell Proliferation/genetics* ; Vimentin/metabolism* ; MicroRNAs/metabolism* ; Colonic Neoplasms/genetics* ; RNA-Binding Proteins/metabolism* ; Gene Expression Regulation, Neoplastic/genetics* ; Cell Movement/genetics*

Silver nanoparticles (AgNPs) is known as one of the most valuable metal nanoparticles in antibacterial and anticancer application. AgNPs-resistant bacteria has been documented, but it is unclear whether cancer cells can also escape the anti-cancer effect of AgNPs. In this study, we aimed to investigate this phenomenon and its underlying mechanism. The antibacterial activity and cytotoxicity of AgNPs were measured in the presence of HeLa cell metabolites. The status of AgNPs in the system associated with metabolites were characterized by UV-Vis, Zetasizer Nano ZS, and transmission electron microscopy. Non-targeted metabolomics was used to reveal the metabolites components that bind with AgNPs. HeLa cells were injected intraperitoneally to establish the tumor-bearing mice model, and the stability of AgNPs in mice serum was analyzed. The results manifested that HeLa cell metabolites inhibited the anticancer and antibacterial effects of AgNPs in a dose-dependent manner by causing AgNPs aggregation. Effective metabolites that inhibited the biological activity of AgNPs were stable in 100 ℃, insoluble in chloroform, containing sulfur elements, and had a molecular weight less than 1 kDa in molecular weight. There were 115 compounds bound with AgNPs. In vitro experiments showed that AgNPs aggregation occurred only when the concentration of α-ketoglutarate (AKG) and glutathione (GSH) together reached a certain threshold. Interestingly, the concentration of AKG and GSH in HeLa cellular metabolites was 10 and 6 times higher than that in normal cervical epithelial cells, respectively, which explained why the threshold was reached. Furthermore, the stability of AgNPs in the serum of tumor-bearing mice decreased by 20% (P < 0.05) compared with the healthy mice. In conclusion, our study demonstrates that HeLa cells escaped the anti-cancer effect of AgNPs through the synergistic effect of AKG and GSH, suggesting the need to develop strategies to overcome this limitation.
Humans ; Animals ; Mice ; HeLa Cells ; Silver/pharmacology* ; Ketoglutaric Acids/pharmacology* ; Metal Nanoparticles ; Anti-Bacterial Agents/pharmacology* ; Glutathione ; Microbial Sensitivity Tests